A cell suspension that is obtained after dissociation of neural tissue comprises a wide variety of different cell types. But, selective targeting and isolation of a special cell type is often the prerequisite for experimental in vitro studies on these cells. Relatively easy techniques like density gradient centrifugation or isolation of living cells by culture conditions lead to low purities and massive cell loss.
A widely used approach in the field of neurobiology is the use of transgenic mice expressing a fluorescent protein controlled by a cell-type specific promoter in combination with fluorescence-activated cell sorting that facilitates isolation of a special cell population (Nolte et al., 2001: Glia 33:72-86; Tomomura et al., 2001: Eur J Neurosci 14:57-63; Tamamaki et al., 2003: J Comp Neurol 467, 60-79; Zuo et al., 2004: J Neurosci 24:10999-11009).
But, besides the need of transgenic mice and expensive equipment this procedure is quite cumbersome and takes several hours. Fluorescence-activated cell sorting of neurons was also described for non-transgenic animals, after labeling the cells with the neuron specific marker NeuN (Guez-Barber et al., 2012: J Neurosci Methods 203, 10-18). Nevertheless, this approach does not allow for the separation of living neurons as it requires labeling of an intracellular marker.
Another technique leading to isolation of neural cells was described by Barres et al. (1988: Neuron 1:791-803). The method named immunopanning uses an antibody mediated cell adhesion. Originally described for the isolation of Retinal cells using Thy-1 as specific marker for retinal ganglion cells, the method was also applied for the isolation of neurons (Cahoy et al., 2008: J Neurosci 28:264-278) by subsequent depletion of oligodendrocytes, microglia and astrocytes. But, an one step isolation for neurons using this technique has not been described so far.
A straightforward method for the isolation of a desired cell type is the magnetic separation of cells, e.g. the magnetic activated cell sorting (MACS technology, Miltenyi Biotec GmbH, Germany; U.S. Pat. No. 5,411,863, U.S. Pat. No. 5,543,289, U.S. Pat. No. 6,020,210, U.S. Pat. No. 6,417,011). This technology requires a marker that allows direct separation of the cells of interest by an antibody coupled to a magnetic microbead. However, a general neuron specific cell surface marker is not known so far and the direct magnetic cell sorting of viable neurons is therefore not applicable. Nevertheless, a negative isolation strategy can be applied to isolate cell populations that cannot be addressed directly. In this approach, non-target cells are magnetically labeled and depleted, thereby isolating the unlabeled cells of interest.
The object of the present invention is to provide an improved method for separating living neuronal cells and removing non-neuronal cells from a cell suspension derived from nervous tissue.
All references, publications, and patent applications disclosed herein are hereby incorporated by reference in their entirety.